CN102368071B - Chemiluminescent immunoassay kit for detecting mycoplasma pneumoniae IgM antibody - Google Patents
Chemiluminescent immunoassay kit for detecting mycoplasma pneumoniae IgM antibody Download PDFInfo
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Abstract
The invention provides a chemiluminescent immunoassay kit for detecting a mycoplasma pneumoniae IgM antibody, which belongs to a medical detection reagent. The chemiluminescent immunoassay kit comprises a monoclonal antibody coated microplate of anti fluorescein isothiocyanate, fluorescein isothiocyanate labeled mycoplasma pneumoniae antigen, horseradish peroxidase labeled anti-human IgM and a chemiluminescent substrate. The method has the advantages of low background, high specificity and strong affinity, simultaneously, effectively reduces the batch difference and has large application value in the immunodetection field.
Description
Technical field
The present invention relates to medical science and detect reagent, particularly a kind of chemiluminescence immunoassay kit that detects mycoplasma pneumoniae IgM antibody.
Background technology
Mycoplasma pneumoniae (Mycoplasma pneumonia, MP) in 1962 in SARS (Severe Acute Respiratory Syndrome) patient sputum separation isolate first, that a class is between bacterium and virus, acellular wall, can be on without life nutrient culture media the procaryote of growth and breeding, belong to protobiont door, Mollicutes, Mycoplasmas, Mycoplasmataceae, Mycoplasma, to cause children's tracheitis, bronchitis, primary atypical pneumonia, community acquired pneumonia (community acquired pneumonia, CAP) important pathogen body, after infection, also can cause encephalitis, myocarditis, immune hemolytic anemia, nervous system damage, kidney damage, the complication such as infectious mononucleosis and Kawasaki disease.
MP propagates with the form of aerosol particles by the spittle, can cause in the world and distribute respiratory tract infection or little popular, at interval of 3-7, an endemic can occur.MP the incidence of infection changes with research object, season, diagnostic method difference.The mankind are to the general susceptible of MP, and wherein 5-20 year is group of people at high risk.According to estimates, approximately have 20-40% infant to infect MP, and in the CAP infant of being in hospital, infection rate is 10-20% in outpatient service infant, CAP infant MP infects positive rate and infant age and increases and be proportionate.The mixed infection of MP and other cause of diseases often makes course of disease delay, is about 10% with streptococcus pneumonia mixed infection rate, is about 8.8% with mycoplasma pneumoniae mixed infection rate.
After mycoplasma pneumoniae infection human body, through the latent period in 2-3 week, performance clinical symptoms.The symptoms such as their early stage has pharyngalgia, headache, heating, weak, DOMS, anorexia, feels sick, vomiting, general medium temperature, after 2-3 days, there is obvious respiratory symptom, outstanding behaviours is paroxysmal irritable cough, to attach most importance to night, cough a small amount of glutinous phlegm or mucus purulent sputum, blood-stained sputum, also can have expiratory dyspnea, pectoralgia sometimes.Except the performance of respiratory system, Eaton agent pneumonia can occur together multisystem, multiple organ injury.Skin lesion can show as maculopapule, erythema nodosum, vesicle etc., gastrointestinal system is vomitted as seen, the infringement of diarrhoea and liver function, the more common hemolytic anemia of hematological, the visible polyradiculitis of central nervous system damage, meningoencephalitis and cerebellar lesion etc., cardiovascular system pathology occasionally has myocarditis and pericarditis.Therefore it is extremely important for finding as early as possible and treat MP infectious diseases that, MP infects early diagnosis.
At present the laboratory diagnostic method of MP is mainly contained the methods such as pathogen isolation culture technique, nucleic acid detection technique, serodiagnosis.Wherein pathogen isolation culture technique due to technical conditions require high, complex operation, required time is long waits restriction, can not solve the needs of clinical quick diagnosis, is replaced gradually by other method for quick.PCR method susceptibility is high, high specificity, is particularly useful for MP Rapid&Early diagnosis.But PCR because of its responsive vulnerable to pollution, be prone to false positive results, and technical requirement is high, needs special instrument and equipment and technician, expensive etc., and reason has limited it in clinical universal development.Enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) have quick, responsive, easy, be easy to the advantages such as standardization, it is developed and widespread use rapidly, become one of detection method of widespread use.
Chemiluminescence immune assay (Chemiluminescence Immunoassay, CLIA) be a kind of highly sensitive microdetermination technology growing up in recent years after radiommunoassay and EIA enzyme immunoassay, there is wide, the easy and simple to handle advantage such as fast of high sensitivity, sensing range, as the replacer of radioimmunoassay, EIA enzyme immunoassay, it is current optimal immune analysis method.Do not see at present based on chemiluminescence immune assay and detect the highly sensitive of mycoplasma pneumoniae, the kit of good stability.
Summary of the invention
The present invention is according to the blank of the existence in above-mentioned field and demand, a kind of kit that detects mycoplasma pneumoniae IgM antibody is provided, experiment showed, to have that Background is low, specificity is high, affinity is strong, effectively reduce differences between batches simultaneously, in field of immunodetection, there is larger using value.
Detect the chemiluminescence immunoassay kit of mycoplasma pneumoniae IgM antibody, the coated solid phase carrier of monoclonal antibody that comprises anti-fluorescein isothiocynate, the mycoplasma pneumoniae antigen of marked by fluorescein isothiocyanate, anti-human IgM and the chemical luminous substrate of horseradish peroxidase-labeled, described chemical luminous substrate comprises substrate solution A and substrate solution B, what wherein A liquid was consists of: pH value is 7.2～8.5, concentration is that in the phosphate buffer of 10～30mM, to add final concentration (W/V) be 0.02～0.1%Proclin300,0.1～1.0% Tween-20 and 10mM urea peroxide solution; B liquid is that in the Tris-HCI damping fluid of 50mM, to add final concentration (W/V) be 0.02～0.1%Proclin300, the 4-iodophenol of 1.0～3.0mM, 0.1%～1% BSA, 0.1～1% cetrimonium bromide, 10～50mM luminol solution.
The coated concentration of the coated microwell plate of monoclonal antibody of described anti-fluorescein isothiocynate is: 2ug/ml.
The coated microwell plate of monoclonal antibody of described anti-fluorescein isothiocynate makes by following steps: the coated microwell plate of anti-fluorescein isothiocynate monoclonal antibody coating buffer that is mixed with 2ug/ml with the carbonate buffer solution that pH value is 9.6.
The mycoplasma pneumoniae antigen of described marked by fluorescein isothiocyanate makes by following steps:
(1) mycoplasma pneumoniae antigen is dialysed under 2～8 ℃ of conditions with 50mmol/L pH 9.6 carbonic acid buffers,
(2) fluorescein isothiocynate is dissolved in dimethyl sulfoxide (DMSO), and final concentration is 1mg/mL,
(3) in mycoplasma pneumoniae antigen: fluorescein isothiocynate is 1mg: the ratio of 150 μ g slowly adds the solution of step (2) in mycoplasma pneumoniae antigenic solution and mixes, 4 ℃ of reactions of lucifuge 8 hours, add NH4Cl to final concentration 50mmol/L, 4 ℃ of cessation reactions 2 hours
(4) marked by fluorescein isothiocyanate antigenic solution is dialysed in the phosphate buffer of 0.05mol/L pH 7.2, add containing 0.1% (W/W) NaN3,1% (W/W) bovine serum albumin(BSA), concentration is in the phosphate buffer of 10mmol/L pH 7.2, and 4 ℃ keep in Dark Place.
Described kit also comprises dilution, cleansing solution, mycoplasma antibody IgM negative control, positive control, calibration object, the anti-human IgM of horseradish peroxidase-labeled.
Described anti-human IgM is that how anti-goat-anti people IgM is, how anti-the anti-human IgM of rabbit is or mouse-anti people IgM monoclonal antibody.
The present invention is with fluorescein isothiocynate (FITC) mark mycoplasma pneumoniae antigen, simultaneously with the coated solid phase carrier of anti-FITC monoclonal antibody, set up indirectly coated mycoplasma pneumoniae chemical luminescence detection method, the method advantage is that Background is low, specificity is high, affinity is strong, effectively reduce differences between batches simultaneously, in field of immunodetection, there is larger using value.
Kit of the present invention adopts the coated microwell plate of FITC monoclonal antibody, form solid phase FITC monoclonal antibody-FITC labelled antigen-antibody complex with mycoplasma pneumoniae IgM in FITC labelled antigen and sample to be tested, form solid phase FITC monoclonal antibody-FITC labelled antigen-antibody-mark two anti-sandwich complex with the anti-human IgM of mark again, then add chemical luminous substrate, utilize Chemiluminescence Apparatus to detect relative luminous intensity, mycoplasma pneumoniae IgM antibody in qualitative or half-quantitative detection sample to be tested, thereby avoided enzyme to be excused from an examination, sensitivity that agent exists is low, the shortcomings such as part substrate is poisonous or carcinogenic, it is fast that this kit has reaction velocity, light signal is not subject to influence of temperature change, cost is low, easily store, specificity is high, product differences between batches are little, the advantage such as convenient for production, can be the acute respiratory disease auxiliary diagnosis that clinical mycoplasma pneumoniae is relevant and provide more special, fast, reliable diagnosis basis.
A main contribution of the present invention has been to provide a kind of new Chemoluminescent substrate formula, compares with adopting the kit of existing conventional substrate solution formula, has the advantages such as highly sensitive, good stability.
Embodiment
Embodiment 1, kit preparation method of the present invention
1, kit of the present invention comprises:
1) mycoplasma pneumoniae IgM positive control; 2) mycoplasma pneumoniae IgM negative control; 3) mycoplasma pneumoniae IgM calibration object; 4) microwell plate of coated FITC monoclonal antibody; 5) the mycoplasma pneumoniae antigen of FITC mark; 6) anti-human IgM enzyme labeling thing; 7) 20 times of concentrated cleaning solutions; And 8) Chemoluminescent substrate.
Wherein, microwell plate is the micropore lath in 24,48 or 96 holes; Described anti-human IgM is that how anti-goat-anti people IgM is, how anti-the anti-human IgM of rabbit is or mouse-anti people IgM monoclonal antibody; Described enzyme horseradish peroxidase.
2, the preparation method of I kit of the present invention:
With mycoplasma pneumoniae IgM positive serum preparation positive control, with Healthy Human Serum preparation negative control, with mycoplasma pneumoniae IgM positive serum preparation calibration object, coated in microporous plate FITC monoclonal antibody, FITC mark mycoplasma pneumoniae antigen, the anti-human IgM of enzyme labeling, preparation chemical luminous substrate, the above-mentioned component of packing, and be assembled into finished product.
The anti-FITC monoclonal antibody of 2.1 coated in microporous plate:
2.1.1 coated
With the carbonate buffer solution that 50mM pH value is 9.6, be mixed with the FITC monoclonal antibody coating buffer that coated concentration is 2ug/ml, and coating buffer is carried on to microwell plate (100ul/ hole), 2-8 ℃ of coated 18-24 hour.
2.1.2 sealing is with containing 5%BSA, and pH value is 7.0-7.5, and the Tris damping fluid that concentration is 10mM seals the solid phase carrier after above-mentioned washing as confining liquid.
The preparation of the anti-human IgM antibody of 2.2 enzyme mark adopts the sodium periodate method of improvement.Concrete steps are referring to " Acta Biochimica et Biophysica Sinica " 16 volume the 6th phase in 1984, Luo Jiali etc. " the some problems in sodium periodate oxidation horseradish peroxidase (HRP) and the labeling method of high effect ".
The preparation of 2.3FITC mark mycoplasma pneumoniae antigen
Mycoplasma pneumoniae (purchased from the expensive Xiang in Guangzhou) 2mg is dialysed under 2～8 ℃ of conditions with 50mmol/L pH 9.6 carbonic acid buffers, FITC is dissolved in dimethyl sulfoxide (DMSO) (DMSO), final concentration is 1mg/mL, in antigen: FITC=1mg: the ratio of 150 μ g slowly adds FITC dimethyl sulphoxide solution in antigenic solution to be marked and mixes, 4 ℃ of dark places reaction 8 hours, add the NH4Cl solution of 5mol/L to final concentration 50mmol/L, 4 ℃ of cessation reactions 2 hours, FITC labelled antigen solution is dialysed limpid to dislysate in the phosphate buffer of 0.05mol/L pH 7.2, add 0.1% (weight) NaN3, in the 10mmol/L phosphate buffer of 1% (weight) bovine serum albumin(BSA) (BSA) pH 7.2, preserve 4 ℃ of dark places.
The preparation of 2.4FITC labelled antigen, enzymic-labelled antibody dilution
The preparation of the anti-human IgM working fluid of 2.5FITC mark mycoplasma antigen and enzyme labeling
The anti-human IgM antibody of FITC labelled antigen, enzyme labeling of preparation is diluted to respectively to variable concentrations with dilution, and use chessboard titrimetry is selected best dilutability labelled antigen, labelled antibody working concentration is (V/V) 1: 3000,1: 6000.
2.620 cleansing solution doubly
2.7HRP chemical luminous substrate
Each 1 bottle of A liquid and B liquid, each 1 bottle of A liquid and B liquid, what wherein A liquid was consists of: pH value is 8.0, and in the phosphate buffer that concentration is 10mM, adding final concentration (V/V) is 0.02%Proclin300,1.0% Tween-20 and 10mM urea peroxide solution; B liquid is that in the Tris-HCI damping fluid of 50mM, to add final concentration (W/V) be 0.02%Proclin300, the 4-iodophenol of 2mM, 0.1% BSA, 0.1% cetrimonium bromide, 10Mm luminol solution.
II kit:
Other is with I kit, the formula of HRP chemical luminous substrate is: each 1 bottle of A liquid and B liquid, what wherein A liquid was consists of: pH value is 8.0, concentration is that in the phosphate buffer of 30mM, to add final concentration (V/V) be 0.05%Proclin300,0.5% Tween-20 and 10mM urea peroxide solution; B liquid is that in the Tris-HCI damping fluid of 50mM, to add final concentration (W/V) be 0.05%Proclin300, the 4-iodophenol of 3mM, 1% BSA, 1% cetrimonium bromide, 50mM luminol solution.
III kit:
Other is with I kit, the formula of HRP chemical luminous substrate is: each 1 bottle of A liquid and B liquid, what wherein A liquid was consists of: pH value is 8.0, concentration is that in the phosphate buffer of 20mM, to add final concentration (V/V) be 0.04%Proclin300,0.1% Tween-20 and 10mM urea peroxide solution; B liquid is that in the Tris-HCI damping fluid of 50mM, to add final concentration (W/V) be 0.04%Proclin300, the 4-iodophenol of 2mM, 0.7% BSA, 0.4% cetrimonium bromide, 30mM luminol solution.
Contrast agents box: other is with I kit, and chemical reflective substrate is commercially available chemical luminous substrate (A liquid is urea peroxide solution, and B liquid is luminol solution, purchased from Huzhou Yingcheng Biological Technology Co., Ltd.).
3, the using method of kit of the present invention
1) in 2-8 ℃ of refrigerator, take out kit, equilibrium at room temperature 15 minutes.
2) take out coated slab, insert on grillage.
3) negative control, negative control, each 2 every hole 50ul in hole of calibration object are established in each test, the sample to be tested 50ul of pre-dilution (1: 100), and then every hole adds 50ul FITC labelled antigen, and vibration mixes rear 37 ℃ of incubations 30 minutes.
4) get rid of dereaction liquid, the cleansing solution after dilution is filled it up with in every hole, washes plate 5 times, finally on clean thieving paper, buckles dry.
5) every hole adds respectively 100 μ L enzyme labeling things, 37 ℃ of incubations 20 minutes.
6) get rid of dereaction liquid, the cleansing solution after dilution is filled it up with in every hole, washes plate 5 times, finally on clean thieving paper, buckles dry.
7) each hole adds each 50 μ L of Chemoluminescent substrate A, B, fully vibrates and mixes, room temperature (20～25 ℃) lucifuge reaction 5 minutes with micro-oscillator.
8) must in 5th～30 minutes after adding Chemoluminescent substrate, measure, on chemiluminescence measuring instrument, sequentially measure the luminous intensity (RLU) in each hole, 1 second/hole of Measuring Time.
9), with calibration object critical value, qualitative or sxemiquantitative judges in sample to be tested whether have mycoplasma pneumoniae IgM antibody or its content
4, the parallel contrast test of kit of the present invention and ELISA reagent
To normal check sample 358 examples, respiratory tract infection sample 254 examples (being derived from Wuzhou treatment and prevention of tumour research institute) adopt kit of the present invention (chemoluminescence method) and mycoplasma pneumoniae IgM antibody test reagent (euzymelinked immunosorbent assay (ELISA)) (kit is purchased from the expensive Xiang in Guangzhou) to carry out Parallel testing, and trace routine and result judgement are carried out in strict accordance with each reagent instructions.
Table 1. mycoplasma pneumoniae IgM antibody test reagent (euzymelinked immunosorbent assay (ELISA)) measurement result
Table 2. kit measurement result of the present invention
Table 3. kit of the present invention and mycoplasma pneumoniae IgM antibody assay kit (euzymelinked immunosorbent assay (ELISA)) comparison
Described in table 1-3, data show, kit of the present invention is 90.4% with the positive coincidence rate of contrast enzyme-linked immunologic detecting kit, and negative match-rate is that the coincidence rate of 99.6%, two kind of method of inspection is 98.2%.Empirical tests, enzyme-linked immunologic detecting kit detects 2 parts of false positives, 9 parts of false negatives, and kit testing result of the present invention conforms to completely with clinical effectiveness.
II kit, III kit contrast test experience result conform to I kit.
Embodiment 2. kit luminous substrate of the present invention and commercially available Chemoluminescent substrate contrast test
Contrast agents box: identical with the kit of embodiment 1, difference is that chemical luminous substrate adopts the Chemoluminescent substrate (purchased from Huzhou Yingcheng Biological Technology Co., Ltd.) of commercially available routine.
It is respiratory tract infection sample 254 examples and Healthy People blood sample in embodiment 1 that step 1. detects sample.
Detect operation steps with the using method of embodiment 1 step 3.Result is as shown in the table.
The chemical luminous substrate of table 4. I kit of the present invention chemical luminous substrate and market sale is compared
Described in table 4, data show, I kit chemical luminous substrate of the present invention is 91.5% with the positive coincidence rate of contrast chemical luminous substrate, and negative match-rate is that the coincidence rate of 100%, two kind of method of inspection is 98.7%.Empirical tests, contrast luminous substrate detects 8 parts of false negatives, and kit testing result of the present invention conforms to completely with clinical effectiveness.
II kit, III kit contrast test experience result conform to I kit, the present invention are described than adopting the kit of contrast luminous substrate and have higher detection sensitivity.
2-8 ℃ of step 2. I kit of the present invention, II kit, III kit were preserved after 6 months, for detection of the same lot sample of step 1 this, result shows, positive rate does not change, the rate that conforms to Clinical results is 100%.And adopt the kit that contrasts luminous substrate, and to preserve after 6 months for 2-8 ℃, false negative ratio brings up to 11/94 by 8/94, illustrates in kit of the present invention that chemical luminous substrate has good stability, is suitable for a large amount of production in batches, and testing result is reliable and stable.
Claims (6)
1. detect the chemiluminescence immunoassay kit of mycoplasma pneumoniae IgM antibody, the coated microwell plate of monoclonal antibody that comprises anti-fluorescein isothiocynate, the mycoplasma pneumoniae antigen of marked by fluorescein isothiocyanate, anti-human IgM and the chemical luminous substrate of horseradish peroxidase-labeled, described chemical luminous substrate comprises substrate solution A and substrate solution B, consisting of of A liquid wherein: pH value is 8.0, concentration is that in the phosphate buffer of 10～30mM, to add final concentration be 0.02～0.05% (W/V) Proclin300,0.1～1.0% (W/V) Tween-20 and 10mM urea peroxide solution; B liquid is that in the Tris-HCl damping fluid of 50mM, to add final concentration be the Proclin300 of 0.02～0.05% (W/V), the 4-iodophenol of 2.0～3.0mM, the BSA of 0.1%～1% (W/V), the cetrimonium bromide of 0.1～1% (W/V), 10～50mM luminol solution.
2. chemiluminescence immunoassay kit according to claim 1, the coated concentration of the coated microwell plate of monoclonal antibody of described anti-fluorescein isothiocynate is: 2 μ g/mL.
3. chemiluminescence immunoassay kit according to claim 1, the coated microwell plate of described anti-fluorescein isothiocynate monoclonal antibody makes by following steps: the coated microwell plate of anti-fluorescein isothiocynate monoclonal antibody coating buffer that is mixed with 2 μ g/mL with the carbonate buffer solution that pH value is 9.6.
4. chemiluminescence immunoassay kit according to claim 1, the mycoplasma pneumoniae antigen of described marked by fluorescein isothiocyanate makes by following steps:
(1) mycoplasma pneumoniae antigen is dialysed under 2～8 ℃ of conditions with 50mmol/L pH9.6 carbonic acid buffer,
(2) fluorescein isothiocynate is dissolved in dimethyl sulfoxide (DMSO), and final concentration is 1mg/mL,
(3), in mycoplasma pneumoniae antigen: fluorescein isothiocynate is that the ratio of 1mg:150 μ g slowly adds the solution of step (2) in mycoplasma pneumoniae antigenic solution and mixes, 4 ℃ of reactions of lucifuge 8 hours, add NH
4cl is to final concentration 50mmol/L, 4 ℃ of cessation reactions 2 hours,
(4) marked by fluorescein isothiocyanate antigenic solution is dialysed in the phosphate buffer of 0.05mol/L pH7.2, adds containing 0.1%(W/W) NaN
3, 1%(W/W) bovine serum albumin(BSA), concentration is in the phosphate buffer of 10mmol/L pH7.2,4 ℃ keep in Dark Place.
5. chemiluminescence immunoassay kit according to claim 1, described kit also comprises dilution, cleansing solution, mycoplasma antibody IgM negative control, mycoplasma antibody IgM positive control, mycoplasma antibody IgM calibration object, the anti-human IgM of horseradish peroxidase-labeled.
6. chemiluminescence immunoassay kit according to claim 1, described anti-human IgM is that how anti-goat-anti people IgM is, how anti-the anti-human IgM of rabbit is or mouse-anti people IgM monoclonal antibodies.
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| CN106248944A (en) * | 2016-06-30 | 2016-12-21 | 深圳市亚辉龙生物科技股份有限公司 | A kind of CPn IgG chemiluminescence immune detection reagent kit and preparation method thereof |
| CN106053440A (en) * | 2016-06-30 | 2016-10-26 | 深圳市亚辉龙生物科技股份有限公司 | Mycoplasma pneumoniae IgG chemiluminescence immunoassay kit and preparation method thereof |
| CN108872571B (en) * | 2017-05-11 | 2023-05-23 | 国家纳米科学中心 | Thioflavine T-based immunoassay method |
| CN109738642A (en) * | 2018-12-28 | 2019-05-10 | 无锡壹闪生物科技有限公司 | The kit and its detection method of dual-antigen sandwich method detection immunoglobulin M |
| CN110286236A (en) * | 2019-07-25 | 2019-09-27 | 科尼格沃斯(无锡)医学科技有限公司 | The detection method of mycoplasma pneumoniae detection kit, preparation method and mycoplasma pneumoniae |
| CN110483644A (en) * | 2019-09-10 | 2019-11-22 | 山东硕景生物科技有限公司 | Positive serum substitute of mycoplasma pneumoniae IgM antibody and preparation method thereof |
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